In primary breast cancers dichotomic classification of E-cadherin expression, according to an arbitrary cutoff, may be inadequate and lead to loss of prognostic significance or contrasting prognostic indications. We aimed to assess the prognostic value of high and low E-cadherin levels in a consecutive case series (204 cases) of unilateral node-negative non-lobular breast cancer patients with a 8-year median follow-up and that did not receive any adjuvant therapy after surgery.Expression of E-cadherin was investigated by immunohistochemistry and assessed according to conventional score . Multiple correspondence analysis was used to visualise associations of both categorical and continuous variables. The impact of E-cadherin expression on patients outcome was evaluated in terms of event-free survival curves by the Kaplan–Meier method and proportional hazard Cox model.Respect to intermediate E-cadherin expression values (2+), high (3+) or low (0 to 1+) E-cadherin expression levels had a negative prognostic impact. In fact, both patients with a low-to-nil (score 0 to 1+) expression level of E-cadherin and patients with a high E-cadherin expression level (score 3+) demonstrated an increased risk of failure =1.71, confidence interval (CI)=0.72–4.06 and HR=4.22, CI=1.406–12.66) and an interesting association with young age.The findings support the evidence that high expression values of E-cadherin are not predictive for a good prognosis and may help to explain conflicting evidence on the prognostic impact of E-cadherin in breast cancer when assessed on dichotomic basis. CDH1 gene with a decreased E-cadherin expression .We considered 234 consecutive patients who underwent surgery for a breast cancer between January 1989 and December 1993 at the Surgical Units of Ferrara S. Anna Hospital University or at Surgical Units of the Ferrara province's hospitals. Informed written consent was obtained from all patients and the study was approved by the University of Ferrara Research Ethics Committee. All patients had a non-lobular cancer and did not receive any adjuvant therapy after surgery because axillary lymph node negative (pN0) at diagnosis. Additional eligible criteria were: pathologic stage T1 to T3, availability of at least 10 resected axillary lymph nodes, absence of synchronous bilateral tumours or any other malignancy before breast cancer diagnosis and up to 6 months after surgery, absence of distant metastases at diagnosis and up to 6 months after surgery, and no neo-adjuvant therapy. Clinical baseline and patients follow-up data  were extracted from the Ferrara Cancer Registry. Data on patient age, tumour histology, pathologic stage, grading, oestrogen receptor (ER) and progesterone receptor status were also collected. After assessment of routine biological markers, for 204 patients , a residThe article was prepared in agreement with the reporting recommendations for tumour marker reporting studies , pH 7.8, to unmask antigenic sites after formalin fixation. IHC was performed with an automated immunostainer . Slides were stained for E-cadherin using the ECH-6 antibody  and Vectastain ABC peroxidase kit  was used to reveal antibody binding. Slides treated with normal serum or isotype-matched antibodies were used as negative controls. Endogenous biotin was saturated with a biotin blocking kit (Vector Laboratories). Two pathologists (MP and PQ) independently examined all TMA sections. For each tumour at least 400 cells were counted, and the percentage of immunostained cells was recorded. E-cadherin expression was scored according to the product between the intensity coefficient  and the frequency of positivity coefficient  and categorised as follows: 0, negative score; 1+, score 1–4; 2+, score 5–8; 3+, score 9–12. TMA blocks were assembled as follows. A true-cut needle  was used to punch 3-mm spaced holes in the recipient block. Donor blocks of formalin-fixed, paraffin-embedded archival primary tumour samples were retrieved after re-evaluation of haematoxylin- and eosin-stained slides. Representative tumour areas were identified, 4 mm diameter cores of tumour tissues were removed from each donor block and transferred in the recipient block (24 samples per slide). Tissue microarray was then incubated for 15 min at 37 °C to allow the tumour cores to firmly adhere to the recipient block. Consecutive 5-χ2 metric, whereas the measure on the axes do not have any physical meaning. To help the interpretation of the results, a cluster analysis was performed using the coordinates of the points, representing tumours, computed by the MCA. The silhouette index  that visualises on a bi-dimensional plot the association of both categorical and continuous variables . For thete index  was usedhttp://www.r-project.org) was utilised throughout this study.As reported in vs E-cadherin 0 was 1.04 (P=0.88), for E-cadherin 2+ vs E-cadherin 1+ was 0.62 (P=0.22) and for E-cadherin 3+ vs E-cadherin 2+ was 2.94 (P=0.03). The results of the multivariable model are reported in vs patients with intermediate E-cadherin expression . Noteworthy, also patients whose tumour expressed high levels of E-cadherin (score 3+) had an unfavourable outcome when compared with patients with E-cadherin 2+ . The prognostic impact of the other variables analyzed, for example, a lower risk of failure associated with ER expression, corresponded to that expected from previous studies.The follow-up of the study was closed on the 31 December 2002. The median follow-up of the 204 patients was 118 months even though it was curtailed at 8 years when only about 12% patients were lost to follow-up. During follow-up, 23 patients developed distant metastases, 14 a local relapse, 8 a contralateral tumour, 8 another malignancy and 37 dead as first event. The Kaplan–Meier survival estimates, according to the different scores of E-cadherin, are shown in Downregulation of the epithelial cell–cell adhesion molecule E-cadherin is frequently associated with neoplastic transformation and progression in breast cancer . HoweverOne of the possible biological explanations of such a finding could be the epithelial–mesenchymal transition, a process in which epithelial cells lose their characteristic polarity, disassemble cell–cell junctions and become more migratory. One of the most peculiar aspects of epithelial–mesenchymal transition is the ‘cadherin isoform switching’ that consists in a downregulation of E-cadherin expression and a concomitant upregulation of N-cadherin with profound effects on cell phenotype and behaviour. In fact, upregulation of N-cadherin has been shown to promote tumour cell motility and invasion (In conclusion, present results indicate that the IHC evaluation of E-cadherin expression in terms of negative/positive status should perhaps be too naive as E-cadherin positivity might actually hide complex biological interactions that may determine the course of tumour progression and disease outcome. A hypothesis that certainly deserves further investigation.


Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was investigated.+ T-Cells were added to the epithelial airway model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array.SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and advanced computer-controlled analysis. Finally, human primary CD4SPP were taken up by epithelial cells, macrophages, and dendritic cells. This uptake coincided with secretion of pro-inflammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific way  and led to a decreased secretion of pro-inflammatory cytokines.These results display a possible mechanism of how SP-D can modulate the inflammatory response to inhaled allergen. Over the past 30 years asthma prevalence has markedly increased. A high percentage of asthmatics is sensitized against pollen . Whole pin vitro-monocultures which lack important cell-to-cell interactions to resemble the in vivo situation. In vivo, cells are able to communicate directly and indirectly with each other in order to orchestrate a specific or unspecific immune response [Phleum pratense) as well as the influence of SP-D in cells within an epithelial airway model, consisting of human monocyte-derived macrophages (MDM), ECs, and human monocyte-derived dendritic cells (MDDC) [+-T-cells were isolated from blood of allergic donors, added to the epithelial airway model, and inflammatory mediators were measured after further incubation.Most of the studies, including the above mentioned, which focused on the interaction of SP-D and allergen particles or other pathogens were accomplished in response -15. To as (MDDC) -18. Thiss (MDDC) . By usinPhleum pratense) pollen were obtained from Allergon . All other reagents, unless otherwise specified, were purchased from Sigma Chemical .Rat recombinant SP-D (SP-D) was purified by maltose affinity chromatography from the media supernatant of cultured Chinese hamster ovary cells stably transfected with a full-length rat SP-D cDNA clone as described previously . TimothyAll subjects who donated blood gave their written consent after being fully informed about the purpose and nature of the studies which were approved by the Ethics Committee of Hannover Medical School.g for 4 min. The supernatant was filtered  and centrifuged twice at 2500 g for 10 min. The resulting pellet was resuspended in 1 ml of sterile NaHCO3 (0.1 M) for fluorescence labelling or phosphate buffered saline (PBS) for direct use in the experiments. To determine the number of SPP, an aliquot was diluted in PBS (1:100) and then counted in an improved Neubauer chamber.SPP were isolated from timothy grass pollen as described previously . 300 mg 9 - 2 × 109 SPP in 1 ml NaHCO3 (0.1 M) was used. The suspension was transferred into the vial of reactive dye and rotated for 1 h at room temperature in the dark. Sterile PBS (14 ml) was added, centrifuged at 2500 g for 12 min and the pellet was resuspended in 1 ml of PBS. The SPP were counted under fluorescence light in an improved Neubauer chamber.Immediately after the isolation procedure, SPP were fluorescently labelled with Alexa Fluor 488 fluorescent dye . For the staining procedure an amount of 1 × 10During this study, measurement of Lipopolysaccharide (LPS) contamination of the SPP was not performed.2.A549 cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 Medium  supplemented with 10% heat-inactivated foetal calf serum (FCS) and 1% penicillin-streptomycin in a 37°C humidified atmosphere with 5% CO2, pores of 3.0 μm in diameter, high pore density PET membranes for 6-well plates; BD Biosciences, Basel, Switzerland). Macrophages and dendritic cells were derived from human blood monocytes as described before [2 humidified atmosphere. Triple cell co-cultures were incubated for 8 hours with 10 × 106Alexa488 labelled SPP which were added to the apical chamber for uptake quantification or with unlabelled SPP for subsequent cytokine/chemokine determination.Cultures were prepared as previously described  and as sd before . Briefly+-T-cells were isolated according to the manufactures instructions  and subsequently stored for 1 week at -80°C. After 8 hours incubation of the triple cell co-cultures with unlabelled SPP and SP-D, thorough washing steps with phosphate buffered saline solution (PBS) were performed. The membrane with the triple cell co-cultures was excised, turned around, and placed in a 12-well plate so that the MDDC resided on the upper side of the membrane  containing 170 mg/mL Mowiol 4-88 .Antibodies were diluted in PBS as follows: mouse anti-human CD14 1:20 , mouse anti-human CD86 1:20 , goat anti-mouse cyanine 5 1:50 , and phalloidin-rhodamine 1:100 .A Zeiss LSM 510 Meta with an inverted Zeiss microscope  was used. Image processing and visualization was done using IMARIS, a 3D multi-channel image processing software for confocal microscopic images .®  as already described for polystyrene particles of different sizes [After image acquisition the total particle number per scan was counted with the particle tracking software Diacountnt sizes ,22 and int sizes . For eac+-T-cells in fresh medium. All cytokines and chemokines were measured by bead-based protein quantification. A Bioplex 200 System  and a Milliplex bead kit  were used according to the manufacturer's instructions. Since the values of Interleukin (IL)-8 were above the limit of quantifications, cell culture supernatants were diluted and IL-8 was measured by enzyme linked immunosorbent assay  according to the manufacturer's instructions. The limit of detection was the lowest standard value of the respective cytokine/chemokine. The limit of quantification was the lowest standard value where the duplicates had a coefficient of variation < 0.2 and a mean which was at least 5 times higher than the blank value.Supernatants were taken after 8 hours incubation with unlabelled SPP and further 72 hour incubation with CD4To assess the effect of SP-D, the percentage of positive cells was determined and the number of SPP per cell was counted. In addition, we calculated an uptake index (UI), which considered the percentage of cells containing particles multiplied by the number of SPP inside single cells. The UI was estimated from 6 individual experiments as described by the following formula:(p): Percentage of cells containing particles. P(c): Number of particles inside cells.C®, Version 4.03. Statistical comparison of the means was performed by ANOVA, followed by a Bonferroni correction. P-values < 0.05 were considered to be significant.Values are given as means ± SEM. Statistical analysis was performed using GraphPad PrismSPP were found intracellulary in MDM Figure , EC Fig, and MDDA detailed quantification revealed that 43.0 ± 9.8% of the MDM took up 21.5 ± 7.2 SPP per cell after 8 hours incubation with 10 million SPP. Furthermore, 3.3 ± 0.7% of the EC took up 2.9 ± 0.3 SPP, and 47.9 ± 13.0% of the MDDC internalized 31.2 ± 18.2 SPP on average Figure . WhereasIn contrast, the number of SPP, which were taken up by individual MDM and MDDC, was lower after co-incubation with SP-D compared to control conditions without SP-D Figure . SP-D (1The uptake index (UI) for SPP in MO after incubation with 10 million SPP, i.e. 685.4 ± 193.2, stayed nearly constant after co-incubation with increasing concentrations of SP-D  that considered both, the percentage of cells containing particles as well as the number of SPP inside single cells, we demonstrated that the total number of SPP stayed constant in MDM, was increased in EC, and was decreased in MDDC. Incubation with SPP increased secretion of (pro-)inflammatory cytokines and chemokines. SP-D inhibited the IL-8 release from the cells.in vitro and in vivo and further emphasize the need of complex in vitro systems which may much better mimic specific in vivo situations. In fact, our results show a decreased UI for the MDDC which would be in line with the results of the study of Winkler and co-workers considering a substantial amount of particle clearance during the longer observation period they used [® does hardly allow for a subjective counting, this should be taken into consideration. Importantly, we were able to reliably determine SPP uptake in contrast to binding and focused only on the intracellular SPP. Recently we showed that SP-D increased the amount of human primary bronchial epithelial cells with attached SPP [Little is known about the uptake of naturally occurring allergen particles by resident lung cells. In addition, although SP-D is one of the first proteins which comes into contact with inhaled particles and thereby modulates immune responses , only fehey used . Within ched SPP . It is fched SPP . In accoched SPP , we founched SPP . Hence wIncubation with 10 million SPP led to an increased (pro-)inflammatory response within the epithelial airway model after further 72 hours incubation with fresh medium compared to cultures without SPP. This clearly indicates an inflammatory effect of SPP that may also occur in the lung after a single inhalation of allergen particles and that may be independent of allergic sensitization to the particles. The increased secretion of several cytokines (e.g. IL-8) determines a pro-inflammatory action of the SPP which was previously observed for various other allergens in different experimental systems -30. InteImportantly, an allogenic response within the co-culture model can be excluded. With respect to IL-8, we observed cytokine secretion after measuring just the cell culture system without any stimulus. However, after incubating the cell-culture system with SPP, we found a significant increase of IL-8. Hence we conclude that this increase is not the result of an allogenic stimulation. The addition of SP-D decreased this SPP-modulated increase of IL-8 which further supports the notion of being no allogenic effect.SP-D led to a significantly decreased level of IL-8 in the supernatant in the cell cultures after incubation. This is in contrast to a former study with primary bronchial epithelial cells in monocultures. It has been observed that epithelial mono-cultures and triple cell co-cultures react differently upon exposed to various particles e.g. diesel exhaust particles, titanium dioxide as well as single-wall carbon nanotubes ,34. We hImportantly, the cell cultures within these experiments were observed under a light microscope after the end of each experiment. Thereby, we could clearly see the normal cell shape with no signs of apoptotic blebbing or disturbance in the confluent A549 cell layer. In addition, our positive control Concanvalin A led to a secretion of most of the measured cytokines and chemokines. From dead cells, the secretion would not be so pronounced.+-T-cells, too -specific T-cells. However, it is important to note that up till now we were only able to cultivate the T-cells within the co-culture model for 72 hours in order to guarantee viability of all cells. Crucial parameters of the immunological responses within the body normally occur over longer time periods. This limitation has to be considered. Thereby, the epithelial airway model should be further optimized so that a standardized use of the four different cell types is warranted and T-cell-dependent mediators and behaviour can be evaluated.T-cell-dependent mediators were measured 72 hours after adding of the CD4Taken together, SPP uptake in various cell types, i.e. MDM, EC and MDDC, of a human epithelial airway model is modulated by SP-D. Thereby, a SPP-induced inflammation is decreased by SP-D. In addition, the contact to other lung cells modulates the A549 cells to react more similar than primary lung cells.EC: Epithelial Cell; MDM: Monocyte derived Macrophage; MDDC: Monocyte derived Dendritic Cell; PSG: Pollen Starch Granules; SPP: Subpollen Particle; SP-D: Surfactat Protein-DThe authors declare that they have no competing interests.CS planned the concept and study design, performed the experiments, interpreted the results and wrote major parts of the manuscript. BMR planned the concept and study design, interpreted the results and wrote major parts of the manuscript; VJE planned the concept and study design and interpreted the results, PG, FB, HDL, MN, AB, and NK made substantial contributions to the analysis and interpretation of the data. JMH planned the concept and study design, made substantial contributions to the analysis and interpretation of the data and wrote major parts of the manuscript. All of the authors have critically read the manuscript and approved its submission.Table S1. Secretion of cytokines and chemokines after 8 hours incubation with subpollen particles (SPP) plus surfactant protein d (SP-D) as well as further 72 h incubation with fresh medium.Click here for file


Loa loa. The survival and development of larvae were monitored.Immunological mechanisms involved in the survival and development of human filarial species in the vertebrate host are poorly known due to the lack of suitable experimental models. In order to understand the role of cytokines in the survival and development of filarial larvae in the vertebrate host, we infected different strains of BALB/c mice deficient in a number of cytokine or cytokine receptor genes with L. loa and necropsies were performed at different time intervals up to 70 days post infection to monitor the survival and development of L. loa larvae. The larvae were teased out of the skin, muscles, peritoneal and pleural cavities, heart and lung tissues. The length and width of the recovered larvae were measured to assess their growth.BALB/c mice genetically deficient in IL-4R, IFN-γ, IFN-γ/IL-5, IL-5, and IL-4R/IL-5 cytokine or cytokine receptor genes were infected with a human strain of In mice deficient for IL-4R, IFN-γ, IFN-γ/IL-5, IL-5 and IL-4R/IL-5, the larvae survived up to 5, 20, 40, 50 and 70 days respectively. Worms recovered 70 days post infection in IL-4R/IL-5 DKO mice were young adults and measured 10.12 mm in length and 0.1 mm in width. Overall, 47% of larvae were recovered from subcutaneous tissues, 40% from muscles, 6% from the peritoneal cavity and 4% from the pleural cavity, lungs and heart.L. loa exhibits a differential survival and development in different strains of cytokine or cytokine receptor gene knockout mice with IL-4R and IL-5 playing critical roles in the host resistance to L. loa infection. The knock out BALB/c mouse therefore represents a useful tool to explore the key effectors of adaptive immunity involved in the killing of the L. loa parasite in a mammal host. L. loa develop severe adverse events (SAEs) following ivermectin treatment [Loiasis is a neglected tropical disease that has recently emerged as a disease of public health importance due to its negative impact on the control of onchocerciasis in areas where the two infections co-exist. Individuals harboring a heavy microfilarial load of reatment . Presentreatment -4. PendiL. loa develops up to patency in baboons and drills. These non human primate models have been used to study the biology of L. loa in the mammal host [L. loa and the host [L. loa does not undergo a full course of development in laboratory wild-type mice. In filariasis, knockout mice have been used as tools for the study of the host immune response [L. loa, it was observed that infective larvae survived for only 1 week in immune-competent BALB/c and Swiss mice, whereas in the same mice immune-depressed with hydrocortisone, L. loa larvae survived for up to 3 weeks [L. loa from the murine host. In order to improve on the knowledge of the biology of L. loa in the mammalian host and to better understand the role of cytokines in the survival and development of L. loa in the mammalian host, we infected 5 strains of BALB/c mice deficient in specific cytokines (or a cytokine receptor) with human L. loa and the survival and development of the larvae were monitored.A major obstacle facing research on loiasis and particularly vaccine development has been the lack of suitable animal models. Apart from humans, mal host -7 and sothe host -11. Howeresponse -15. In e 3 weeks . This inThe mice used in the experiment were obtained from the Institute of Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Germany, where the following strains had been used and described before :-/- mice, originally purchased from The Jackson Laboratory ; (ii) BALB/c IL-5-/- mice [-/-/IL-5-/- mice; (iv) BALB/c IL-4Rα-/-mice [-/-/IL-5-/-, which were provided by K.I.M [(i) BALB/c IFN-γ-/- mice , (iii) Bα-/-mice  and (v) by K.I.M  , maintained at 23 - 28°C and 79% - 80% relative humidity. The flies were then dissected in RPMI 1640 medium (Sigma) and the infective larvae collected, counted and concentrated in 100 - 200 μl RPMI 1640. The mice were each inoculated with 100 L3s subcutaneously in the right lumbar area.Infected animals were dissected at different time intervals . The protocol used in the dissection of mice was described in previous studies ,16. AnimChrysops were measured and served as a baseline to calculate larval growth.The length and width of larvae recovered from different strains at different time points were measured using a microscope fitted with a Camera Lucida. L3s dissected from The mean number of larvae recovered at different necropsy time was generated and a two way analysis of variance (ANOVA) was used to compare the mean recovery at different time points in different strains. The proportions of larvae recovered from each organ were generated and compared using the Chi-square test. The Benferroni Post Hoc test was used to compare the increase in length and width of larvae recovered in different strains of mice at different time points after log transformation to normalize the data. All the statistical tests were performed at a 5% significance level.-/-/IL-5-/- DKO supported the infection better than any other strain (70 days), followed by IL-5-/- KO (50 days), IFN-γ-/-/IL-5-/- DKO (40 days), IFN-γ-/- KO (20 days) and lastly IL-4R-/- KO (5 days). Within each mouse strain, a significant difference (IL-5-/- KO (P < 0.001), IFN-γ-/- (P < 0.001), IFN-γ-/-/IL-5-/- (P < 0.05) and IL-4R-/-/IL-5-/- (P < 0.05)) was observed in the mean number of larvae recovered at different time points.In all the five strains investigated, the number of larvae recovered dropped with time Figure . A drast-/-/IL-5-/- DKO mice, there was a significant difference (p < 0.05) between the proportion of larvae recovered from the subcutaneous tissue and the muscles. In IL-4R-/-/IL-5-/- DKO and IL-4R-/- KO mice, more larvae were recovered from the muscles, whereas in the other strains of mice more larvae were recovered from the subcutaneous tissue.In both single and double cytokine/cytokine receptor knockout mice, larvae were recovered from the subcutaneous tissues, muscles, peritoneal and pleural cavities, heart and lungs. More than 80% of larvae were recovered from the muscle and subcutaneous tissues in all strains of mice Table . With th-/- KO, day 10 in IFN-γ-/-/IL-5-/- DKO and IL-5-/- KO and day 15 in IL-4R-/-/IL-5-/- DKO  to muscles, and internal organs. Larvae were found in the peritoneal and pleural cavities two days after inoculation. Larvae reached the heart and the lungs by day 5 in IFN-γKO Table . In IFN--/- KO mice that kept the infection only for a few days (data not included in statistics below), larvae increased significantly (p < 0.001) in length and width with time , 40 days (IFN-γ-/-/IL-5-/- DKO), 20 days (IFN-γ-/- KO) and 5 days (IL-4R-/- KO). This differential survival and development of L. loa in cytokine knockout BALB/c mice emphasizes the importance of these classical Th1 and Th2 cytokines in the development and establishment of filarial worms in their vertebrate hosts. Even though wild type laboratory mice do not support the life cycle of human filariae, this study shows the importance of genetically modified mice (knockout mice) as experimental models to study the immune effectors regulating the survival and establishment of L. loa in a mammalian host.The fact that small laboratory animals are not permissive to Monanema martini infection in the striped grass mouse Lemniscomys striatus and also for Litomosoides sigmodontis in BALB/c mice and the jird, Meriones unguiculatus [Monanema and Litomosoides experimental models.In all strains of BALB/c mice used in this study, we observed a massive clearance of larvae a few days after infection. More than 50% of them were killed within the first 2 days of infection. The rapid clearance of filarial larvae inoculated into the mammalian host was first reported in iculatus -24. Thisiculatus -24. In tL. loa larvae is similar to what was observed in other studies with L. loa using immunodepressed BALB/c and Swiss mice, and with L. sigmodontis and Brugia malayi in Cotton rat and jird hosts respectively [Migrating larvae were found in the genital organs, peritoneal and pleural cavities, lungs and heart. This migration and localization of ectively ,25.L. loa reaches patency in the non-human primate 4 to 7 months post inoculation [L. loa is achievable in some knockout mice, however, it would be necessary to keep the worms for longer periods in these mice to observe further morphological changes, possible maturation and patency.The measurement of the length and width of larvae recovered at various time points post inoculation showed a significant increase with time, indicating that the larvae effectively grew and developed. Larval growth was accelerated 20 to 30 days post infection. Larvae recovered after 30 days showed a 2 to 3-fold increase in length and width. The worms recovered 70 days post infection were immature adults and were not mature enough to produce microfilariae. culation . The datL. loa worms in the mammalian host. As was observed in previous studies [L. loa in the heart and lungs indicates that they may have used the thoracic duct to migrate to those organs as was demonstrated in M. martini in its natural host L. striatus, L. sigmodontis and B. malayi in their surrogate host Meriones unguiculatus [L. loa could occur in the human host, this might be associated with clinical manifestations such as the tropical pulmonary eosinophilia that is not presently known to be related to loiasis.In all strains of mice, a large number of larvae (about 80%) remained in the subcutaneous tissue and the muscles, which are the sites of predilection of  studies ,25, onlyiculatus ,21,24. IL. loa. When IL-5 was singly knocked out, larvae survived up to 50 days. But when IL-5 and IL-4R were both knocked out, the larvae survived up to 70 days, suggesting an additive effect of IL-4R on IL-5 in the clearance of L. loa worms. These two cytokines have been previously associated with resistance of the host to infection. IL-4 and IL-5 in addition to IL-13 are required to prevent the development of adult worms as well as development of microfilaremia [L. loa compared to singly knocked out IL-5. This study clearly demonstrates that IL-5 is an important effector in the clearance of L. loa worms, the other cytokines (IFN-γ and IL-4) play a secondary role. IL-5 alone has been shown to control adult worm development in primary infection. In murine infection with L. sigmodontis, its deficiency leads to an increased parasite burden [Onchocerca lienalis infective larvae in mice [L. sigmodontis model, it was also shown that the depletion of IL-5 leads to higher worm recovery and a higher level of microfilaraemia [In this study, we observed that the double absence of IL-4R and IL-5 promoted the survival of ilaremia ,27. Whene burden ,28,29. I in mice . In thoslaraemia .L. loa exhibits a differential survival and development in different strains of cytokine/cytokine receptor knockout BALB/c mice with the IL4R/IL-5 DKO accommodating the worms for longer period and provide better conditions for their growth. Findings of this study using the knockout mice approach have confirmed previous findings on the role of some cytokines in preventing this filarial infection in mice. This therefore opens an avenue for the exploitation of this approach to accelerate investigations to better understand the immune effectors important for the survival and development of L. loa in the mammalian host.The data obtained shows that IMMIP: Institute of Medical Microbiology, Immunology and Parasitology; SAE: Severe adverse events; KO: Knockout; RPMI: Roswell Park Memorial Institute medium1640; ANOVA: Analysis of variance; DKO: Double knockout; EFINTD: European Foundation Initiative for Neglected Tropical Diseases; EPIAF: Enhanced Protective Immunity against FilariasisThe authors declare that they have no competing interests.NT participated in the study design, carried out laboratory experiments, analyzed the data and drafted the manuscript. SW participated in the study design, interpreted the results and edited the manuscript. JCN participated in production of infective larvae and dissection of mice. MEE participated in the production of infective larvae and dissection of mice. SS participated in the interpretation of the results and edited the manuscript. PE participated in the study design, and edited the manuscript. KIM: interpreted and edited the manuscript. AH participated in the study design, interpreted the results and edited the manuscript. All authors read and approved the final version of the manuscript.


MicroRNAs are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in the control of developmental and physiological processes. In particular, the developing brain contains an impressive diversity of microRNAs. Most microRNA expression profiling studies have been performed in human or rodents and relatively limited knowledge exists in other mammalian species. The domestic pig is considered to be an excellent, alternate, large mammal model for human-related neurological studies, due to its similarity in both brain development and the growth curve when compared to humans. Considering these similarities, studies examining microRNA expression during porcine brain development could potentially be used to predict the expression profile and role of microRNAs in the human brain.MicroRNA expression profiling by use of microRNA microarrays and qPCR was performed on the porcine developing brain. Our results show that microRNA expression is regulated in a developmentally stage-specific, as well as a tissue-specific manner. Numerous developmental stage or tissue-specific microRNAs including, miR-17, miR-18a, miR-29c, miR-106a, miR-135a and b, miR-221 and miR-222 were found by microarray analysis. Expression profiles of selected candidates were confirmed by qPCR.The differential expression of specific microRNAs in fetal versus postnatal samples suggests that they likely play an important role in the regulation of developmental and physiological processes during brain development. The data presented here supports the notion that microRNAs act as post-transcriptional switches which may regulate gene expression when required. Mus musculus mature miRNAs and 1240 represent mature miRNAs from Homo sapiens. At present, only 211 mature miRNAs are annotated for Sus scrofa in miRBase 16.0, and more than half of these are in silico predictions. In the last three years, many studies on identification and characterization of porcine miRNAs have been published. A number of studies are based on cloning and sequencing MicroRNAs (miRNAs) are small (approximately 22 nucleotides long), highly conserved, non-coding RNA molecules that modulate gene expression at the post-transcriptional level by binding to their target mRNAs The abundance and diversity of miRNAs varies in different tissues. A number of miRNAs are expressed in highly tissue- or stage-specific patterns Various techniques, such as miRNA cloning, fluorescent in situ hybridization (FISH), northern blot, microarray, deep-sequencing and quantitative real-time PCR (qPCR), have been successfully used for miRNA investigation. Currently, microarrays and deep sequencing are the most commonly used techniques for high-throughput profiling of miRNA expression. Quantitative real-time PCR is an extremely sensitive technique that enables validation of selected candidate miRNAs. Both microarray and qPCR can benefit from the Locked Nucleic Acid (LNA) technology, which increases the thermal stability of the oligonucleotides The majority of the reported miRNA research has been focused on disease-related studies in either human or rodents. However, there is a need to examine miRNAs further in other mammals, such as the domestic pig. The pig brain shows more similarity to the human brain, with respect to anatomy, size, growth and development, compared to brains of other laboratory animals In this study, expression of miRNAs in the pig developing brain was investigated by microarray analysis and subsequently validated by qPCR. Expression profiling of two distinct brain regions was performed. These regions were acquired from three different developmental stages: gestation day 50 (F50), gestation day 100 (F100) and three month-old postnatal brain (termed adult). We selected these three particular stages of development, for the following reasons: At F50, the cerebellum and cortex are in an intensive neural differentiation stage The normalization and data filtering resulted in 1088 high quality probe signals, which represented both human and porcine miRNAs. In the initial analysis of the data, we identified general similarities and differences between the samples by means of principal component analysis (PCA). The analysis of the microarray data in −05. A number of miRNAs including the entire miR-17/92 cluster (with miR-17 and miR-18a being particularly significant), as well as the miR-106a/363 cluster, excluding miR-19b-2 and miR-92a-2 , exhibited decreasing expression throughout development. The second group, e.g.; the miR-29 family (containing miR-29a –b –c) and miR-22, miR-24, miR-27b and miR-142-5p showed increasing expression with age progression, with a particularly high expression in the adult tissues. The two most interesting miRNAs within this group include the miR-221/222 cluster. This cluster had high expression within the F50 cortex and F100 cortex, compared to the adult (where high expression is detected mostly in the cerebellum). Interestingly, miR-455-3p and miR-455-5p were highly expressed in both the F100 cerebellum and the adult cortex, which suggests that the same miRNAs are expressed in both regions, but seem to reach a maximum level of expression at different time points during development. An interesting expression pattern is represented by miR-216a, miR-216b and miR-217 which were found to be exclusively expressed in the F100 cerebellum, implying that they are highly stage- and tissue-specific miRNAs. These three particular miRNAs represent very promising biomarker candidates . Furthermore, these miRNAs might be involved in control of neurogenesis, which peaks just prior to birth. During the early postnatal period, neurons undergo terminal differentiation. Therefore, it is plausible that miRNAs prominently expressed at this time (for instance miR-24 and the miR-29 family members) might regulate this process. The cerebellum starts to develop very early in fetal life. Therefore, miRNAs up-regulated in F50 cerebellum , miR-18a(2.62E−09), miR-29c(4.71E−09), miR-106a(1.84E−08), miR-135a(8.26E−09), miR-135b(1.99E−08), miR-221(9.19E−05), miR-222(2.04E−05) .−17 to 2.02E−10 which indicated that the results were statistically significant  for both these miRNAs. In addition, miR-135b was highly expressed in the adult cerebellum. Interestingly miR-135a and miR-135b have previously been described in prostate cancer et al., The last two miRNAs: miR-221 and miR-222, which belong to the same cluster, constitute the fourth group of expression pattern observed. Similar to the cluster described above, these two miRNAs exhibited decreasing expression during development in the cortex and increasing expression during development in the cerebellum. It has previously been described that the miR-221/222 cluster shares a similar expression pattern in the adult hypothalamus The overall expression profiling results identified a number of miRNAs that could be important for region-, or stage-specific functions within the brain. Moreover, we have revealed an interesting and novel expression pattern of the miR-135a and miR-135b cluster in the brain.2 = 0.81 to R2 = 0.95. The correlation coefficient values (R2) for particular miRNAs include: miR-17 (R2 = 0.84), miR-18a (R2 = 0.94), miR-29c (R2 = 0.92), miR-106a (R2 = 0.81) and miR-135a (R2 = 0.89) miR-135b (R2 = 0.95), miR-221 (R2 = 0.91) and miR-222 (R2 = 0.88). Thus, the overall correlation between microarray and qPCR results was extremely high. These results strengthen our reported outcomes compared to other studies, where a rather poor degree of correlation has been reported for some miRNAs A comparison between microarray probe intensities and qPCR fold changes indicated that different miRNAs had different levels of correlation, varying from RTargetScan was used to identify putative targets for selected miRNA candidate. In order to make the search more specific, only the most significant gene targets were taken into consideration. The same predicted targets were found for miR-17 and miR-106a. This is due to a high sequence similarity of these two miRNAs. One of the interesting targets included the myelin transcription factor 1-like (MYT1L) gene, which is considered to play a role in the development of neurons and oligodendroglia in the CNS. MiR-18a is predicted to target another CNS related gene, i.e. the neural precursor cell expressed developmentally down-regulated protein 9 (NEDD9). NEDD9 is a docking protein which plays a central co-ordinating role for tyrosine-kinase-based signalling related to cell adhesion. Interestingly, miR-135b is predicted to target a leucin zipper putative tumor suppressor 1 gene (LZTS1). This protein coding gene is ubiquitously expressed in normal tissues and is known to be involved in the cell cycle. Finally, the miR-221/222 cluster targets cyclin-dependent kinase inhibitor 1B (CDKN1B) and the tumor suppressor gene p27Kip1. The miR-221/222 cluster has previously been described to act as a regulator of p27Kip1 We have been able to identify potential developmental and stage-specific miRNA candidates, which are associated with particular functions during neuronal development and differentiation. The expression profiles established in this study provide new knowledge about gene regulation during the cerebellum and cortex development and may be valuable in comparative studies of human brain development. Additionally, we have identified the miR-135a -135b cluster in brain, which has not been previously described. These miRNAs represent potentially interesting candidates for future studies in the brain. Additional functional studies are however, required, to assess the precise role of the miRNA candidates identified.Two tissues were selected in this study, including the cortex and cerebellum. These regions were acquired from three different developmental stages: gestation day 50 (F50), gestation day 100 (F100) and three month-old postnatal brain (termed adult). Sampled tissues were immediately snap frozen in liquid nitrogen and stored at −80°C until used. For each developmental stage, four animals were used. Two replicates from each animal were submitted for RNA isolation, resulting in eight samples representing a particular tissue, at a particular developmental stage. The pigs included in this study were raised under production conditions according to Danish standards for animal husbandry. Since the animals were not subjected to experimental procedures, approval was not necessary. The pigs were euthanized by a licensed veterinarian.The small RNA fraction was isolated from the individual tissues using the miRVana™ miRNA Isolation Kit .100–180 mg of tissue was processed per sample, according to the manufacturer's recommendations. The resulting samples were DNAse treated with the DNA-free™ Kit . The quantity and the quality were analyzed on a NanoDrop 1000 spectrophotometer  and by visual inspection of the agarose gel electrophoresis images. Additionally, the integrities of the samples were measured by Small RNA Assay on a 2100 Bioanalyzer .250 ng of the small RNA fraction was used for each sample. Three biological replicates for each developmental stage/tissue were used for the microarray study. Samples were labeled using MiRCURY™ LNA miRNA Power labeling Kit , following the manufacturers' recommendations. Spike-ins (used as control probes) were added in equal amounts to each reaction and labeled.Samples were hybridized to Version 11.0 arrays annotated to miRBase version 15.0, which have 1970 unique capture probes. The hybridization was performed according to the miRCURY™ LNA array manual using a Tecan HS4800 hybridization station . After hybridization, the microarray slides were scanned and stored in an ozone- free environment (ozone level below 2.0 ppb), in order to prevent potential bleaching of the fluorescent dyes. The miRCURY™ LNA array microarray slides were scanned using the Agilent G2565BA Microarray Scanner System  and the image analysis was carried out using the ImaGene 8.0 software .The text files, generated by Imagene v.8.0, were imported into Rosetta Resolver and normalized . Data were filtered by using probes showing a standard deviation (SD) above 0.1. The final, filtered data set consisted of intensity values for 1088 probes. Data are MIAME compliant and the raw data are deposited in the GEO database with accession number of: (GSE24106).All clustering and statistical analysis was performed in TMeV −04 was used.All 1088 filtered probes were used as input for a two-way ANOVA with age and brain regions used as factors. The levels for age were F50, F100 and adult, while the levels for the brain region were the cerebellum and the cortex. The full list of miRNA probes can be found in the GEO database where microarray data are submitted with an accession number of: GSE24106. The analysis was performed in TMeV and a significance cutoff p-value of less than 1.00E−04  which is a medium stringent significance filter was implemented based on our experience with miRNA array profiling, biological knowledge and the genomic context of miRNAs. For instance, the interesting candidate miR-221 which is a cluster member with miR-222 would have been discarded by using more stringent cutoff of. 1.00E−05 .There are inherently false positive discoveries in array studies. The selected cutoff of 1.00EProbes were first annotated to pig (ssc), and following this, other significant probes were annotated against human (hsa), mouse (mmu) and rat (rno). Some probes have multiple capture probes, as optimal probes are available for multiple species.Synthesis of cDNA for miRNA qPCR was performed as a one tube reaction where polyA tailing and reverse transcription was conducted simultaneously . Two technical replicates were used for reverse transcription, resulting in 16 cDNA samples for each particular stage and tissue. Briefly, 10 ng of the small RNA fractions was used for cDNA synthesis in a 10 µl reaction. The resulting cDNA was diluted 8 times and 1 µl was used for each qPCR reaction. Each miRNA candidate was evaluated in a total panel of 96 samples.Eight different candidate miRNAs: hsa-miR-17, hsa-miR-18a, hsa-miR-29c, hsa-miR-106a, hsa-miR-135a hsa-miR-135b, hsa-miR-221 hsa-miR-222 and two reference miRNAs: hsa-miR-103 and hsa-miR-191 were profiled by qPCR. The choice of candidate genes was based on the statistical significance obtained following an ANOVA test of the microarray results (see results section). The choice of reference genes was based on recommendation from the literature One qPCR reaction per sample for each miRNA candidate was performed, using 5 µl of QuantiFast SYBR Green PCR master mix , 0.25 mM of each primer, 1 µl of 8x diluted cDNA, milliQ water up to 10 µl total reaction volume. PCR amplification was performed in white PCR plates ; on an MX3000P machine . The cycling conditions were: 1 cycle at 95°C/10 min, followed by 40 two-segment cycles of amplification . Mx3000/Mx Pro software was used to construct a melting curve. Standard curves with 5 fold dilutions , were performed for each assay and the PCR efficiency calculations were based on the slopes of the standard curves. Quantification cycle (Cq) and initial template quantities for each reaction were determined with use of the baseline adjustment method from the Mx3000/MxPro software .Quantities of each sample were used for the analysis. GeNorm software was used to analyze the stability of the reference genes: miR-103 and miR-191. An M value below 1.5, which represents gene stability, was considered as acceptable. The two reference genes, namely miR-191 and miR-103 , were used for calculation of normalization factors (NF). Samples were normalized by the NF and fold changes were calculated in relation to the lowest, normalized expressed value, and then tested for normal distribution by GraphPad InStat 3 software. The one factor Analysis of Variance (ANOVA) statistical test was performed to test for significant differences between the means of the analyzed groups Click here for additional data file.Table S1The results of Tukey-Kramer multiple comparisons test between each sample group representing particular developmental stage and tissue. One-way ANOVA was applied to determine the probability values presented.(0.02 MB XLS)Click here for additional data file.Table S2The list of probability values following ANOVA analysis of the microarray data, resulted in a number of microRNA (miRNA) candidates chosen for qPCR validation. P-values for miR-221/222 were obtained when both stage and tissue was considered statistically variable. Values for other miRNAs were obtained when the developmental stage was the considered variable. Ssc and hsa stand for Sus scrofa and Homo sapiens probe, respectively.(0.02 MB XLS)Click here for additional data file.


Cow's milk allergy is one of the most common food allergies among younger children. We investigated IgE antibodies to milk, and IgE and IgG4 antibodies to casein, α-lactalbumin and β-lactoglobulin in cow's milk allergic (CMA) and non-allergic (non-CMA) children in order to study their clinical usefulness.®. For the sIgG4 analysis, 28 atopic and 31 non-atopic control children were additionally included .Eighty-three children with suspected milk allergy  were diagnosed as CMA (n = 61) or non-CMA (n = 22) based on an open milk challenge or convincing clinical history. Their serum concentrations of allergen-specific (s) IgE and IgG4 antibodies were measured using ImmunoCAPA/L corresponding to 100% specificity. All but one of the CMA children aged > 5 years had casein-sIgE levels > 6.6 kUA/L. The non-CMA group had significantly higher sIgG4 levels against all three milk allergens compared to the CMA group. This was most pronounced for casein-sIgG4 in non-CMA children without history of previous milk allergy. These children had significantly higher casein-sIgG4 levels compared to any other group, including the non-milk sensitized control children.The CMA group had significantly higher levels of milk-, casein- and β-lactoglobulin-sIgE antibodies as compared to the non-CMA group. The casein test showed the best discriminating performance with a clinical decision point of 6.6 kUHigh levels of casein-sIgE antibodies are strongly associated with milk allergy in children and might be associated with prolonged allergy. Elevated casein-sIgG4 levels in milk-sensitized individuals on normal diet indicate a modified Th2 response. However, the protective role of IgG4 antibodies in milk allergy is unclear. Food allergies, described as adverse immune responses to food, are common and have increased in prevalence during the past decades. About 5% of the young children and 3-4% of the adults are affected today . Milk, eThe most important allergens in cow's milk are α-lactalbumin , β-lactoglobulin (Bos d 5) and casein (Bos d 8) ,9. Milk Diagnosis and management of food allergy include steps like initial avoidance of the suspected food allergen, skin prick testing and measuring of serum levels of food-specific IgE antibodies mostly using extract-based tests. In time, in order to confirm the diagnosis or to determine whether a reintroduction of the particular food is safe due to tolerance development, oral food challenges could be conducted . Other dHere, in order to study the clinical usefulness of specific antibodies in milk allergy, the concentrations of IgE antibodies to milk, and IgE and IgG4 antibodies to casein, α-lactalbumin and β-lactoglobulin were studied in sera from milk allergic and milk tolerant children.in vitro test (n = 81), or had a history of positive skin prick test to milk (n = 2). Most of them suffered from atopic dermatitis (85%) and some also from asthma (32%). The patients were divided into two groups on the basis of their immediate reactions to an open oral milk provocation challenge test or through their case history. The cow's milk allergic (CMA) group (n = 61) had either a positive challenge result for milk (n = 34) or a convincing history of present milk allergy making a challenge test redundant (n = 27). The non-CMA group (n = 22) contained children who had no allergic reactions to milk at the time of examination. A subgroup of them had obtained tolerance after a previous diagnosed milk allergy  which was verified with a negative challenge test with milk. The remaining children had never had a milk allergy diagnosis . Further details about the study groups are found in Table Eighty-three children with a suspected IgE-mediated cow's milk allergy (CMA) were enrolled in the study. The patients  ranged in age from 0.8 to 15.8 years (median: 3.5 years). All were milk sensitized as revealed by specific IgE 4 analyses, a non-milk sensitized control group was included consisting of children that consulted our allergy clinic due to eczema, urticaria, bronchial asthma or any suspicion of allergic diseases and were examined for the following specific IgE antibodies; milk, Dermatophagoides pteronyssinus, cat dander, egg white, wheat, orchard grass, and Japanese cedar. Those who had no clinical history of milk allergy and without milk-specific IgE antibodies (< 0.35 kUA/L) were selected, and subsequently divided into the following two subgroups. One subgroup consisted of 31 non-atopic controls . They had low total IgE levels  and were negative to all of the specific IgE antibodies examined. The other subgroup consisted of 28 atopic controls . They were sensitized to at least one of the allergens described above, except milk, and had a median total IgE level of 928 kU/L .For the specific IgGInformed consent was obtained from patients, their parents, or both. The study was approved by the Ethics Committee of Fujita Health University School of Medicine.Open oral milk provocation tests were carried out as described in a Japanese guideline . The chi® . The commercially available tests and reagents were used according to the instructions from the manufacturer.Serum levels of total IgE, IgE antibodies to milk, casein, α-lactalbumin and β-lactoglobulin, and IgG4 antibodies to casein, α-lactalbumin and β-lactoglobulin were measured using ImmunoCAPA/L) were assigned a value of 0.34 kUA/L, and all values above 100 kUA/L (higher limit of quantitation) were assigned a value of 101 kUA/L. In the same way, all specific IgG4 levels below the assay cut off (0.07 mgA/L) were assigned a value of 0.06 mgA/L.The Mann-Whitney U-test (two-tailed) was used for comparisons between the groups and p values < 0.05 were considered significant. Receiver operating characteristic (ROC) analysis was performed for the different milk allergen ImmunoCAP tests used for specific IgE measurement . Before A/L), but had a history of positive skin prick tests with wheal diameters of 12 × 4 and 15 × 9 mm, respectively.No significant differences in total IgE levels between the CMA and non-CMA groups were seen Table , while tA/L resulting in a 98% sensitivity for the casein ImmunoCAP test using the traditional assay cut off. The levels of β-lactoglobulin-specific IgE were also increased in the CMA group, but no differences between the groups were seen for α-lactalbumin-specific IgE. There were 19 patients without measurable β-lactoglobulin-specific IgE (< 0.35 kUA/L) and 30 patients without α-lactalbumin-specific IgE, resulting in clinical sensitivity values of 69% for the β-lactoglobulin test and 51% for the α-lactalbumin test, respectively  and without atopy (NAC subgroup) as defined by their sensitization to common inhalant and/or food allergens.The non-CMA group had significantly increased levels of specific IgG4 antibodies to all three milk allergens as compared to the CMA group (p < 0.01 and < 0.001). However, when studying the Tolerant and Negative subgroups of the non-CMA group individually, it was shown that the increased levels of specific IgG4 were seen mainly in the Negative subgroup composed of children without previous history of milk allergy Table .Increased levels of specific IgG4 to α-lactalbumin- and β-lactoglobulin were also seen in the non-milk sensitized control group. However, increased levels of casein-specific IgG4 antibodies, as compared to the CMA group, were only found in the AC subgroup Table . The oveA/L). A majority of them (72%) had casein-specific IgE antibodies above our suggested clinical decision point of 6.6 kUA/L, while all non-CMA children were below this cut-off point for casein-specific IgE.In the present study, it was shown that the levels of IgE antibodies to milk, casein and β-lactoglobulin were increased in Japanese children with milk allergy compared to milk-sensitized children without present symptoms to milk (non-CMA children). When comparing different ImmunoCAP tests for measurement of IgE antibodies to milk, casein, α-lactalbumin and β-lactoglobulin, the best performance in the diagnosis of milk allergy was shown for the casein test. All but one of the CMA children had casein-specific IgE antibodies (> 0.35 kUMilk allergy is an obvious health problem and burden for the individual allergic child. But the disease also causes limitations to the daily life of the immediate family in their effort of trying to avoid accidental exposure of their child to milk. A recent study showed that allergic reactions to accidental exposure to milk allergens indeed are frequent . AvoidinA/L for IgE antibodies to milk and 9 kUA/L for IgE to casein, respectively [Double-blind, placebo-controlled food challenge is considered to be the gold standard for food allergy diagnosis, although it carries some risks for the patient and is time-consuming to conduct . For sevectively . Thus, aectively . For exaectively ,22, someA/L. Also others have shown that patients with prolonged milk allergy generally have higher levels of milk-specific IgE than patients whose allergy has resolved [Studies have shown that milk-specific IgE levels are lower in children who later become tolerant than in those with prolonged allergy, showing that IgE antibody measurements can be used to predict tolerance development ,23,24. Iresolved ,8, and tresolved ,23 and iresolved .The production of IgG4 antibodies is considered to be a normal physiological response to the ingestion of cow's milk . PreviouInterestingly, the Negative subgroup had the highest levels of casein-specific IgG4 of all groups in the study including the AC group and NAC group with non-milk sensitized children. It suggests that casein-specific IgG4 antibodies might be markers of a so-called modified Th2 response as suggested by Platts-Mills . Thus, tA/L, while all non-CMA children were below this cut-off point. It was also shown that the levels of casein-specific IgE remained high in CMA children also after five years of age. We therefore conclude that high levels of casein-specific IgE antibodies are strongly associated with milk allergy in children and might be associated with a prolonged allergy. Results from the present study indicate that high levels of casein-specific IgG4 antibodies are associated with tolerance in milk-sensitized children, but only in subjects on normal milk-containing diet. The protective role of IgG4 antibodies in a so-called modified Th2 response is unclear, and it has not yet been proven that measurement of IgG4 antibodies has a role in the clinical management of food allergy. Thus, more studies are needed before proposing routine use of specific IgG4 measurements in milk allergy.IgE antibody levels to milk, casein and β-lactoglobulin were increased in Japanese children with cow's milk allergy. The casein ImmunoCAP test showed the best clinical performance. A majority of the CMA children had casein-specific IgE levels above our suggested positive decision point of 6.6 kUThe authors declare that they have no competing interests.All authors have contributed to the final interpretation of data and the writing of the manuscript that has been approved by all parts. Furthermore, KI initiated and coordinated the Japanese part of the study. MF carried out sample collection. RM reviewed the results and coordinated the writing of the manuscript. AK coordinated the collaboration between Japan and Sweden. TK performed data analysis. TS supervised the study design and process. MB planned the Swedish part of the study including writing of manuscript.


C. albicans in the arthritic joint altered the local cytokine environment, leading to enhanced Th17 expansion and resulting in a more destructive arthritis.Fungal components have been shown very effective in generating Th17 responses. We investigated whether exposure to a minute amount of Streptococcus pyogenes (SCW) cell wall fragments into the knee joint of C57Bl/6 mice, alone or in combination with the yeast of C. albicans or Zymosan A. During the chronic phase of the arthritis, the cytokine levels, mRNA expression and histopathological analysis of the joints were performed. To investigate the phenotype of the IL-17 producing T-cells, synovial cells were isolated and analyzed by flowcytometry.Chronic SCW arthritis was induced by repeated injection with C. albicans on top of the SCW injection both resulted in enhanced joint swelling and inflammation compared to the normal SCW group. However, only the addition of C. albicans during SCW arthritis resulted in severe chondrocyte death and enhanced destruction of cartilage and bone. Additionally, exposure to C. albicans led to increased IL-17 in the arthritic joint, which was accompanied by an increased synovial mRNA expression of T-bet and RORγT. Moreover, the C. albicans-injected mice had significantly more Th17 cells in the synovium, of which a large population also produced IFN-γ.Intra-articular injection of either Zymosan A or C. albicans, are very potent in interfering with the local cytokine environment in an arthritic joint, thereby polarizing arthritis towards a more destructive phenotype.This study clearly shows that minute amounts of fungal components, like  Rheumatoid arthritis (RA) is a systemic joint disease with an unknown etiology, characterized by a chronic inflammatory infiltration of the synovial membrane, and associated with destruction of cartilage and bone. Both genetic and environmental factors contribute to the development of disease. Various infectious agents, such as bacteria and viruses have long been associated with the pathogenesis of RA Microorganisms are recognized by pattern recognition receptors (PRR), such as Toll-like receptors (TLRs) and C-type lectins, which are essential components of the innate immune system and form the bridge with adaptive immunity. If the inflammatory response is not adequate and/or the infection is not cleared properly, persistence can eventually result in chronic or even auto-immune inflammation Candida albicans is the most common opportunistic fungal pathogen in humans. Infection with C. albicans induces IL-17 producing T helper (Th17) cells in vitro and in vivo in naïve mice C. albicans or Candida-derived β-glucan have been shown to induce and aggravate various models of arthritis C. albicans to skew the T-cell balance in the chronic murine SCW model. This model initiates as a local TNF-dependent macrophage-driven inflammation, at which repeated antigen exposure results in a chronic T-cell dependent arthritic process C. albicans or Zymosan A (<10% of mass) was added to the cell wall fragments of Streptococcus pyogenes (SCW) that were repeatedly injected into the knee joint. During the chronic phase of the arthritis, the development of macroscopic joint swelling and histopathological changes in synovium, cartilage, and bone were determined. Furthermore, the levels of antibodies, secretion of T-cell cytokines and presence of T-cells were examined.The purpose of the present study was to demonstrate the ability of ad libitum. The mice used were between 10–12 weeks of age. All animal procedures were approved by the animal ethics committee of the Radboud University Nijmegen.Male C57Bl/6 mice were purchased from Janvier, France. The mice were housed in filter-top cages; water and food were provided Steptococcus pyogenes T12 organisms were cultured and prepared as described previously Candida albicans (ATCC MYA-3573 (UC 820)) were used Saccharomyces Cerevisiae) was purchased from Sigma-Aldrich and prepared as described earlier 5 particles of C. albicans (1*105≈1 µg) or 2 µg Zymosan, in 7 µl phosphate buffered saline (PBS) into the right knee joint of naive mice. As a control, additional groups were injected with the fungal particles alone. On day 22, twenty-four hours after the last injection, a subgroup of mice was sacrificed for the collection of synovial washouts. Accordingly, patellae with surrounding soft tissue were isolated from inflamed knee joints and cultured 1 hour at RT in RPMI-1640 medium containing 0.1% BSA (200 µl/patella). In addition, the draining lymph nodes  were collected and cells were isolated. Then, 1*105 cells were stimulated for 72 hours with 2 µg/ml plate bound anti-CD3 (R&D systems) and 2 µg/ml plate bound anti-CD28 (BD Biosciences). Thereafter, supernatants were collected, centrifuged and stored for cytokine determination. On day 28, during the chronic joint inflammation, the sera from the remaining mice were collected, the mice were sacrificed, and knee joints were prepared for histology.99 mTc in the inflamed joint due to increased blood flow and edema. Therefore, 0.74 MBq of 99 mTc in 200 µl of saline was injected subcutaneously. After several minutes of distribution throughout the body, external gamma radiation in the knee joints was measured. Swelling was expressed as the ratio of gamma counts in the right (inflamed) knee joint to gamma counts in the left (control) knee joint. Values higher than 1.1 counts per minute were considered to represent joint swelling.Joint swelling was assessed by measuring the accumulation of For standard histological assessment, the isolated joints were fixed for 4 days in 10% formalin, decalcified in 5% formic acid, and the specimens were processed for paraffin embedding. Tissue sections were stained with hematoxylin and eosin. The severity of inflammation in the joints was scored on a scale of 0–3 . Bone destruction was graded on a scale of 0–3, ranging from no damage to the complete loss of bone structure. Proteoglycan (PG) depletion was determined using Safranin O staining. Loss of proteoglycans was scored on a scale of 0–3, ranging from normal, fully stained cartilage to destained cartilage, fully depleted of PG. The scoring of the sections was performed in a blinded manner.Cytokine levels were measured using Luminex multi-analyte technology in combination with Bio-Plex cytokine kits  and performed according to manufacturer's instructions.−ΔCt), for which the expression in the SCW injected group was arbitrarily set to 1. All primer pairs were developed using Primer Express 2.0 (Applied Biosystems) and validated according to protocol.RNA was isolated from synovial knee biopsies using TRI-reagent (Sigma) and treated with DNase to remove genomic DNA. The RNA was subsequently reverse transcribed with oligo(dT) primers in a reverse transcriptase procedure. Quantitative real-time PCR was performed with cDNA specific primers (Biolegio) and SYBR Green PCR Master Mix (Applied Biosystems). The ΔCt method was used to normalize transcripts to GAPDH and to calculate relative mRNA expression  for 30 minutes at 37°C. Next, a 70 µm nylon cell strainer  was used to process the digested tissue. The cell preparation was collected in RPMI with 10% FCS. To isolate the single mononuclear cells, Lympholyte-M (Cederlane) was used according to manufacturer's protocol. All cells were cultured in RPMI-1640  supplemented with 10% FCS. Subsequently, the cells were prepared for intracellular flowcytometry.Synovial cells were stimulated for 5 hours with PMA  and ionomycine  in the presence of Golgiplug (BD Biosciences) according to manufacturers protocol. After staining the cells extracellularly with anti-CD3 APC (BD Biosciences), the cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences). Subsequently, they were intracellularly stained with anti-IFN-γ PE (BD Biosciences) and anti-IL-17 FITC (BD Biosciences). Samples were measured on a FACS Calibur and data were analyzed using FlowJo software.Levels of anti-SCW antibodies in the serum of mice with chronic SCW-induced arthritis (day 28) were analyzed according to standard methods P-values less than 0.05 were considered significant.Results are expressed as the mean ± SEM. Differences between experimental groups were tested using Mann-Whitney U-test or one-way analysis of variance with Dunnett's multiple comparison test, as appropriate. C. albicans to effectively alter the local synovial inflammatory process, heat-killed conidia of C.albicans were co-injected with 25 µg SCW fragments in the knee joint on day 0, 7, 14 and 21. In preliminary experiments, exposure to differential doses of C. albicans to murine peritoneal macrophages resulted in the production of various innate cytokines like TNF-α, IL-1β and IL-6 . We therefore increased the dose of C. Albicans to 1*105 on top of the SCW dose of 25 µg. For comparison, we injected 2 µg Zymosan A, a glucan derived from the yeast cell wall of Saccharomyces Cerevisiae, known for its adjuvant properties C. albicans or Zymosan A alone.Repeated intra-articular injection of SCW bacterial fragments induce arthritic flares that shift from a predominantly macrophage-driven acute inflammation to a T-cell driven chronic inflammation and IL-6 . SubsequC. albicans or Zymosan A alone did not develop joint swelling after the injections  was not significantly enhanced, RANKL was upregulated in the synovium in both the  erosion .C. albicans had been co-injected with SCW into the joints. These findings show that a small amount of C. albicans is sufficient to exacerbate the chronic murine SCW arthritis model by increasing the cartilage and bone destruction.To support that the increase in cartilage destruction was induced by a matrix metalloproteinase–mediated process, we examined the expression of the MMP-specific aggrecan neo-epitope VDIPEN by immunohistochemistry. Repeated SCW injections were sufficient to induce VDIPEN expression at the cartilage sites . The expC. albicans or Zymosan A.To study modification of the adaptive immune responses, we initially studied the B-cell compartment and determined the levels of anti-SCW antibodies of total IgG, IgG1 and IgG3 in the sera of the mice. We previously demonstrated that in the chronic SCW model, antigen-specific antibodies are induced after the fourth i.a. injection of SCW fragments C. albicans  and GATA3 (Th2) mRNA expression levels were not significantly different between the SCW-arthritis groups . In lineand IFNy . Here weC. albicans in the chronic SCW model affected the phenotype of the infiltrated T-cells. On day 22, at the peak of the inflammation, we dissected the inflamed synovium and isolated the residing cells. Intracellular cytokine staining revealed an increased percentage of single IL-17 producing T-cells  and C-type lectin family members  C. albicans on skewing T-cell responses during arthritis, we investigated the chronic SCW model. In this model, chronic T cell dependent arthritis is induced by repetitive intra-articular injection of Steptococcus pyogenes cell wall (SCW) fragments S. pyogenes is a common Gram-positive bacteria that can induce severe rheumatic fever and/or reactive arthritis, depending on the host response C. albicans, would alter the macrophage-derived cytokine production during the flares, aggravating the chronic disease.To explore the potency of a minute amount of C. albicans induce pro-inflammatory cytokine production, like TNF-α, IL-1 and IL-6 C. albicans or Zymosan did not significantly accelerate the model, showing that the co-exposure to C. albicans did not increase the innate immune response like a classical adjuvant Monocytes and macrophages stimulated with C. albicans exposure C. albicans might have altered the antigen presenting function of B-cells. The presence of autoantibodies, like rheumatoid factor (RF) and anti-citrullinated petide antibodies (ACPA) in the sera of patients are early predictors of disease and correlate with arthritis severity This was further strengthened by the fact that we observed no changes in classes of SCW-specific immunoglobulin production. These SCW specific antibodies can be detected in sera during the late chronic stage, and are also TNF-α dependent C. albicans or Zymosan A. Interestingly, although the increase in joint swelling and inflammatory cell influx in the C. albicans co-injected mice was comparable to Zymosan A co-injected mice, the chondrocyte death, bone erosion and cartilage erosion were significantly elevated by C. albicans. Additional analysis revealed that during the chronic phase of the model, the C. albicans exposure contributed to the increased MMP-mediated cartilage destruction and increased bone erosion, suggesting that additional processes were involved in the C. albicans exposed joints. This increase in joint destruction was accompanied by local synovial IL-17 expression, which is thought to/may enhance the role of TNF-α in the progression of the destructive process C. albicans co-injected group. Together, we concluded that the low levels of C. albicans and Zymosan A did not accelerate the model during the first three weeks of repeated antigen injections, but addition of C. albicans did alter the T cell balance and arthritis outcome at the end of the experiment.At day 22, one day after the last injection, when the model shifts from a predominately macrophage driven model to a T cell dependent model, we observed a significantly increased joint swelling in the Candida co-injected group, with a trend for increase in the Zymosan co-injected mice. Furthermore, during the late chronic phase of the model (at day 28) we observed a prolonged increase in joint swelling after co-exposure to both Focusing on the T-cell cytokine production, we observed an increased Th17/Th1 cytokine profile. In line with the increase in the transcriptional factors T-bet and RORγT, this increase was related to an increase in IL-17 single and double positive T-cells. Furthermore, over 90% of the total IL-17 producing cells were CD3+ (data not shown). This supports that T lymphocytes are the main source of IL-17 in this model, rather than innate immune cells capable of producing IL-17 T helper cells are defined T lymphocytes expressing CD4 glycoprotein on their cell surface. We chose CD3 staining over CD4 staining due to the reported sensitivity of CD4 to cleavage during enzymatic digestion of the studied tissue C. albicans does not only increase the ‘classical’ IL-17 producing T-cells, but also promotes the transition to a Th1/Th17 mixed phenotype. The role and induction of Th1 profile versus Th17 profiles in models have received increased attention. In human RA, IFN-γ producing T helper cells are found at the level of the synovial tissue rather than IL-17 producing T helper cells in vivo, that shift towards IFN-γ producing cells during chronic autoimmune inflammation Apparently, in vivo during arthritis will lead to new strategies for the development of novel therapeutic interventions in the Rheumatic diseases.On the other hand, recent studies have shown an inverse relationship between Th17 cells and regulatory T cells C. albicans is sufficient to shift the T-cell balance of the synovial immune response towards a more pronounced Th17/Th1 profile, thereby aggravating the model towards increased cartilage and bone destruction. In the past others have shown that fungal particles, like C. albicans, can accelerate or aggravate arthritic disease. This is the first report showing that this aggravation involves the induction of Th17 cells in vivo. C. albicans is a common commensal that can cause mucosal and systemic infections C. albicans can contribute to the progression of joint destruction in RA remains to be elucidated.In conclusion, exposure to a small amount of the fungal pathogen Figure S1Candida albicans and Zymosan A induce a different cytokine profile in vitro. 1*105 peritoneal macrophages from naive C57Bl/6 mice were stimulated with different concentrations Zymosan or C. Albicans for 16 hours (n=5). Levels of IL-1β, IL-6, IL-10 and TNF-α were determined by Luminex in the supernatants. Results are mean ± SEM.(TIF)Click here for additional data file.